Aplicação da Toxicologia Analítica Verde no desenvolvimento de metodologias baseadas em LC-MS para detecção de Novas Substâncias Psicoativas em amostras biológicas / Application of Green Analytical Toxicology to the development of LC-MS-based methodologies for the detection of New Psychoactive Substances in biological samples

No início dos anos 2000, as Novas Substâncias Psicoativas (NPS) emergiram de forma sem precedentes causando uma drástica mudança no mercado de drogas sintéticas mundial. Estas substâncias são sintetizadas para fins ilícitos e mimetizam o efeito psicoativo das drogas tradicionais. Até o momento, mais de 1000 substâncias foram reportadas mundialmente, representando um grande problema de saúde pública principalmente associado ao desconhecimento das suas propriedades toxicológicas. Por este motivo, métodos analíticos para detectar e quantificar estas substâncias em materiais biológicos são importantes nos casos de toxicologia analítica e forense. Contudo, a tendência de reduzir o impacto ambiental destas metodologias tem ganhado popularidade com a Toxicologia Analítica Verde (GAT). Portanto, o objetivo do presente trabalho foi desenvolver novas técnicas analíticas para analisar as principais classes de NPS em amostras biológicas enquanto aplicando os princípios sustentáveis estabelecidos pela GAT. Os resultados obtidos no presente trabalho são apresentados como coletânea de artigos científicos publicados em revistas. Estes estão descritos nos capítulos 4 a 8. No capítulo 4, uma revisão sobre os desafios no desenvolvimento de técnicas de preparo de amostra para fins forenses é abordada com foco no uso das matrizes secas. No Capítulo 5, está descrito a aplicação da microextração líquido-líquido dispersiva para catinonas sintéticas em amostras de sangue total e urina. No capítulo 6, o artigo descreve o desenvolvimento da técnica microextração líquido-líquido homogênea com solventes de hidrofilicidade comutável para canabinoides sintéticos em amostras de plasma. No capítulo 7, a microextração em fase líquida em placas de 96 poços, cunhada extração paralela em membranas artificiais líquidas, foi desenvolvida para diferentes classes de drogas de abuso, incluindo NPS. O capítulo 8 mostra o desenvolvimento de uma extração por eletromembrana também no formato de placa de 96 poços para catinonas sintéticas em amostras de sangue total. Em todos os trabalhos, as técnicas de extração foram desenvolvidas, otimizadas e validadas. Os princípios da GAT foram aplicados de diferentes formas, como reduzindo o volume de amostra, simplificando os procedimentos, evitando o uso de solventes orgânicos, dentre outros. Assim, alternativas mais sustentáveis para a análise de drogas de abuso em amostras biológicas foram apresentadas e estas ajudam a consolidar e difundir o conceito do desenvolvimento de métodos analíticos com consciência ambiental além de fornecer ferramentas para auxiliar o controle das NPS no país

In the early 2000s, New Psychoactive Substances (NPS) emerged and unprecedentedly changed the illicit drug market. These substances are synthesized for illicit purposes and mimic the psychoactive effect of traditional drugs of abuse. To date, more than 1000 substances have been reported worldwide, representing a major public health problem mainly associated with their mostly unknown toxicological properties. In this context, analytical methods able to detect and quantitate these new drugs in biological specimens are important in cases of analytical and forensic toxicology. However, reducing the environmental impact of these methodologies has recently gained popularity with Green Analytical Toxicology (GAT). Therefore, the aim of this work was to develop new analytical techniques to analyze the main classes of NPS in biological samples while applying the environmentally friendly principles established by GAT. The results obtained throughout the development of the present work were split into four papers (chapters 4-8). In chapter 4, a review of common challenges faced during the development of new sample preparation techniques for forensic applications is described focusing on the use of dried matrices. In chapter 5, the application of dispersive liquid-liquid microextraction for synthetic cathinones in whole blood and urine samples is described. In chapter 6, the application of the somewhat recent switchable hydrophilicity solvent-based homogenous liquidliquid microextraction to synthetic cannabinoids in plasma samples is reported. In chapter 7, liquid-phase microextraction in the 96-well plate format, termed parallel artificial liquid membrane extraction, for different classes of drugs of abuse, including NPS, in plasma samples is presented. In chapter 8, an electromembrane extraction in the 96-well plate format for synthetic cathinones in whole blood samples was developed. In this work, sample preparation techniques were developed, optimized and validated. The principles of sustainable chemistry in method development were applied in different ways, such as reducing the sample volume, simplifying procedures, avoiding the use of organic solvents, among others. Thus, greener alternatives were presented for the analysis of drugs of abuse in biological samples and contribute to consolidate and spread this trend of environmental consciousness during method development. Additionally, valuable techniques that can be used in the combat against NPS were provided

Drogas emergentes: detección mediante sensores electroquímicos

La cantidad de nuevas sustancias psicoactivas (NSP) aumenta en el mercado global cada año. El uso de tecnologías para la producción y la comercialización de las sustancias sintéticas y una demanda exigente de efectos psicotrópicos más potentes son algunos de los factores que contribuyen al incremento de una oferta más variada, así como a la cantidad de consumidores de algún tipo de droga. El desarrollo de herramientas analíticas fiables con capacidad de respuesta in situ para una evaluación preliminar contribuye, significativamente, con la generación de información para atender casos de intoxicaciones, aplicar correctamente la legislación de los países e, inclusive, aportar datos reales para una reclasificación más objetiva de las drogas basada en criterios científicos. Los sensores electroquímicos han ganado terreno en áreas de las ciencias forenses como la toxicología. El uso de tecnologías especializadas para crear electrodos miniaturizados con modificaciones, principalmente con materiales nanoestructurados, guía el desarrollo de dispositivos portátiles de fácil manipulación, altamente versátiles y que requieren micro-cantidades de la muestra. Asimismo, algunos sensores electroquímicos presentan parámetros de desempeño -como límites de detección (LOD) de hasta 0,608 pg/mL, sensibilidad y precisión- que son analíticamente comparables, bajo ciertas condiciones, con las metodologías convencionales acopladas con la espectrometría de masas, las cuales muestran LOD del orden de magnitud entre 10-9 g/mL y 10-12 g/mL para la determinación de algunas drogas. Esta revisión se enfoca en la aplicación y comparación analítica de los sensores electroquímicos voltamperométricos para la determinación de bencilpiperazina (BZP), meta-clorofenilpiperazina (mCPP), 25B-NBO-Me, dimetiltriptamina (DMT), mefedrona (4-MMC), 4-metiletcatinona (4-MEC) y fentanilo (FYL) en representación de las drogas emergentes que se pueden conseguir actualmente en el mercado de las sustancias psicoactivas.

The number of new psychoactive substances (NPS) is increasing on the drug global market every year. New technologies for the production and marketing of synthetic substances, along with a rigorous demand for more powerful psychotropic effects, are some of the factors that contribute to the increase in a more varied supply, as well as the number of drug users. The development of reliable analytical tools with an on-site response capacity for a preliminary determination contributes significantly to the generation of data for the proper care of situations such as poisoning, the correct application of the country's legislation, and even the provision of objective criteria for the reclassification of the substances. Electrochemical sensors have gained ground in areas of forensic science. The use of specialized technologies to manufacture miniaturized electrodes based on modifications, mainly with nanostructured materials, guides the development of versatile easy-to-handle portable devices that require microquantities of the sample. Likewise, some electrochemical sensors have performance parameters such as detection limits (LOD) up to 0.608 pg/mL, sensitivity and precision that are analytically comparable, under certain conditions, with conventional methodologies coupled with mass detection, which show LOD of the order of magnitude between 10-9 g/mL and 10-12 g/mL for drug determination. This review focuses on the application and comparison of voltammetric electrochemical sensors for the determination of benzylpiperazine (BZP), meta-chlorophenylpiperazine (mCPP), 25B-NBO-Me, dimethyltryptamine (DMT), mephedrone (4-MMC), 4-methylethcathinone (4-MEC) and fentanyl (FYL) representing the emerging drugs available to date on the psychoactive substances market.

A quantidade de novas substâncias psicoativas (NSP) sintéticas aumenta a cada ano no mercado global. A utilização de novas tecnologias de produção e comercialização de substâncias, combinado com uma demanda exigente de efeitos psicotrópicos mais potentes, são alguns dos fatores que contribuem para o aumento de uma oferta mais ampla, assim como a quantidade dos números de consumidores de algum tipo de droga. O desenvolvimento de ferramentas analíticas confiáveis e com uma capacidade de resposta in situ para uma avaliação preliminar, contribuem significativamente para a geração de informações científicas para cuidar casos de envenenamento, aplicar corretamente a lei, e até fornecer dados reais para uma reclassificação mais objetiva de drogas com base em critérios científicos. Sensores eletroquímicos ganharam espaço em áreas da ciência forense. A utilização de tecnologias especializadas para criação de eletrodos miniaturizados com modificações, principalmente com materiais nanoestruturados, norteiam o desenvolvimento de dispositivos portáteis altamente versáteis, de fácil manuseio e que necessitam de microquantidades da amostra. Da mesma forma, alguns sensores eletroquímicos apresentam parâmetros de desempenho -como limites de detecção (LOD) até 0.608 pg/mL, sensibilidade e precisão- que são analiticamente comparáveis, sob certas condições, com metodologias convencionais com detecção de massa -mostram LOD de a ordem de grandeza entre 10-9g/mL e 10-12g/mL para a determinação de determinados medicamentos-. Esta revisão tem como foco a aplicação e comparação analítica de sensores eletro-químicos voltamétricos para determinação de benzilpiperazina (BZP), meta-clorofenilpiperazina (mCPP), 25B-NBOMe, dimetiltriptamina (DMT), mefedrona (4-MMC), 4-metilethcatinona (4-MEC) e fentanilo (FYL), que representam os drogas emergentes disponíveis até o momento no mercado de substâncias psicoativas.

Applications of nuclear magnetic resonance spectroscopy in the screening of new psychoactive substances / 中国药科大学学报

@#GC-MS and LC-MS are the main techniques used for the structural identification of new psychoactive substances at present. However, they are hard to give accurate structure information because of the hardly available corresponding reference standards and the quickly changing status of these compounds. This leads tremendous obstacle on the rapid identification of new psychoactive substances. Nuclear magnetic resonance spectroscopy is one of the most effective methods for structures identification. Therefore, NMR is especially suitable for the analysis and identification of new psychoactive substances even with rapid structural changes. This article summarizes the NMR applications for the structural analysis of new psychoactive substances including synthetic cannabinoids, synthetic cathinones, piperazines, phenethylamines, ketamine & phencyclidine-type substances, and fentanyls. It is found that the NMR signals of the main frame structure of each kind of the new psychoactive substances are basically the same. Hence, these frame structure NMR signals can provide scientific evidence for the rapid identification of new psychoactive substances. This article also look ahead the prospect for the application of LC-NMR and DOSY in new psychoactive substances, which provides new ideas for the screening of new psychoactive substances.

New psychoactive substances abuse among patients with access to methadone maintenance treatment in Jiangsu province: a case-control study / 中华流行病学杂志

Objective To explore the reasons and factors associated with new psychoactive substances abuse among patients with access to methadone maintenance treatment (MMT).Methods A well-developed questionnaire and urine tests were used to collect information about demographic characteristics,condition of MMT and drug abuse,family and social support of MMT clients.A 1 ∶ 1 matched case-control study was conducted,and conditional logistic regression model was used to identify factors associated with new psychoactive substances abuse.Results A total of 212 (106 pairs) clients receiving MMT were recruited,and most of them were males (78.3％,166/212),married or cohabitant (48.6％,103/212) and unemployed (63.2％,134/212).The average age of the clients was (45.1 ± 7.2) years.The main types of abused new psychoactive substances were benzodiazepine (62.3％,66/106) and methamphetamine (39.6％,42/106).The proportion of abusing multi new psychoactive substances was 8.5％ (9/106).Results from multivariate conditional logistic regression analysis indicated that using opioid drug during the past 6 months of MMT treatment might increase the risk of abusing new psychoactive substances (OR=3.25,95％CI:1.35-7.79),benzodiazepine (OR=3.25,95％CI:1.11-9.47) and methamphetamine (OR =13.31,95 ％ CI:1.12-158.01).Moreover,MMT for more than 9 years reduced the risk of abuse of new psychoactive substances (OR=0.03,95％ CI:0.01-0.21),benzodiazepine (OR=0.02,95％ CI:0.00-0.36) and methamphetamine (OR=0.02,95％ CI:0.00-0.69).Conclusion Less new psychoactive substances abuse might be associated with longer duration of MMT treatment.And inappropriate support from family and friends might increase the risk of abusing new psychoactive substances in MMT clients,especially in clients who used opioid.

Current situation on new psychoactive substances abuse among methadone maintenance treatment patients in China / 中华流行病学杂志

Methadone maintenance treatment (MMT) greatly contributed to the successful outcomes of prevention and control on both AIDS and drug abuse in China.However,the features on drug abuse changed in the past decades,and the prevalence of new psychoactive substances abuse potentially somehow offset the achievement of MMT.This paper concised the information on research and surveys of this issue that targeting on the current situation,characteristics,related factors and relevant public health problem on new psychoactive substances abuse,among patients who have been on MMT,in China.

New psychoactive substances abuse among patients with access to methadone maintenance treatment in Jiangsu province: a case-control study / 中华流行病学杂志

Objective To explore the reasons and factors associated with new psychoactive substances abuse among patients with access to methadone maintenance treatment (MMT).Methods A well-developed questionnaire and urine tests were used to collect information about demographic characteristics,condition of MMT and drug abuse,family and social support of MMT clients.A 1 ∶ 1 matched case-control study was conducted,and conditional logistic regression model was used to identify factors associated with new psychoactive substances abuse.Results A total of 212 (106 pairs) clients receiving MMT were recruited,and most of them were males (78.3％,166/212),married or cohabitant (48.6％,103/212) and unemployed (63.2％,134/212).The average age of the clients was (45.1 ± 7.2) years.The main types of abused new psychoactive substances were benzodiazepine (62.3％,66/106) and methamphetamine (39.6％,42/106).The proportion of abusing multi new psychoactive substances was 8.5％ (9/106).Results from multivariate conditional logistic regression analysis indicated that using opioid drug during the past 6 months of MMT treatment might increase the risk of abusing new psychoactive substances (OR=3.25,95％CI:1.35-7.79),benzodiazepine (OR=3.25,95％CI:1.11-9.47) and methamphetamine (OR =13.31,95 ％ CI:1.12-158.01).Moreover,MMT for more than 9 years reduced the risk of abuse of new psychoactive substances (OR=0.03,95％ CI:0.01-0.21),benzodiazepine (OR=0.02,95％ CI:0.00-0.36) and methamphetamine (OR=0.02,95％ CI:0.00-0.69).Conclusion Less new psychoactive substances abuse might be associated with longer duration of MMT treatment.And inappropriate support from family and friends might increase the risk of abusing new psychoactive substances in MMT clients,especially in clients who used opioid.

Current situation on new psychoactive substances abuse among methadone maintenance treatment patients in China / 中华流行病学杂志

Methadone maintenance treatment (MMT) greatly contributed to the successful outcomes of prevention and control on both AIDS and drug abuse in China.However,the features on drug abuse changed in the past decades,and the prevalence of new psychoactive substances abuse potentially somehow offset the achievement of MMT.This paper concised the information on research and surveys of this issue that targeting on the current situation,characteristics,related factors and relevant public health problem on new psychoactive substances abuse,among patients who have been on MMT,in China.

Determinação de club dugs em sangue total por cromatografia líquida acoplada a espectrometria de massas com analisador hí­brido quadrupolo-tempo de voo (LC-QTOF-MS) / Determination of club drugs in whole blood by liquid chromatography coupled with mass spectrometry with hybrid quadrupole time-of-flight mass analyzer (LC-QTOF)

As chamadas club drugs compreendem um vasto grupo de substâncias frequentemente utilizadas em bares, festas e raves, com a finalidade de intensificar o contato social e a estimulação sensorial. Englobam desde substâncias sintéticas comumente conhecidas, como a anfetamina, a metanfetamina, o MDMA, até moléculas de surgimento mais recente, denominadas novas substâncias psicoativas. Isoladas ou associadas a outras drogas, é possível que sejam causa de morte per se, ou que predisponham o usuário a envolver-se em situações potencialmente fatais, sendo necessário que os órgãos de Perícia Criminal (Institutos Médico Legais e Institutos de Criminalística) estejam aptos a detectar e quantificar essas substâncias em amostras biológicas. O presente trabalho teve como objetivo desenvolver um método analítico para identificação e quantificação de club drugs em sangue total, utilizando cromatografia líquida acoplada a espectrometria de massas com analisador híbrido quadrupolotempo de voo (LC-QTOF). Após o desenvolvimento do método, este foi validado utilizando as diretrizes do guia de validação do Scientific Working Group for Forensic Toxicology (SWGTOX), sendo analisados de linearidade, limite de detecção, limite de quantificação, efeito matriz, precisão intradia, precisão interdia, exatidão e integridade de diluição, além de recuperação e eficiência do processo. O método desenvolvido compreendeu a determinação de MDA, MDMA, 2C-B, DOB, cetamina, mCPP, cocaína e cocaetileno. Amostras provenientes de casos reais de morte não natural, oriundas do Instituto Médico Legal Aristoclides Teixeira de Goiânia - GO foram analisadas pelo método desenvolvido. 56 casos foram selecionados, em sua maioria com histórico de morte por projétil de arma de fogo e acidente de transito. Das 56 amostras analisadas, 28,5% (n=16) foram positivas para cocaína e/ou cocaetileno. As demais substâncias pesquisadas não foram encontradas nas amostras

Club drugs are a large group of substances consumed in pubs, parties and raves, aiming to intensify social contact and sensorial stimulation. The term comprises largely known substances such as amphetamine, methamphetamine, 3,4-methylenodioxymethamphetamine (MDMA), as well as so-called new psychoactive substances, which are synthetic drugs recently developed or recently introduced in drug market. Club drugs can be taken alone, combined with each other or, most frequently, with alcohol or other commonly abused drugs such as cocaine. In any of these situations, club drugs can possibly be the cause of death or potentialize the involvement of the user with crime and potentially fatal behavior. Thus, official organisms in charge of criminal investigation must be capable of identifying and quantifying these substances in biological samples. The present work aimed the development of an analytical method to identify and quantify club drugs in whole blood, using liquid chromatography - mass spectrometry with hybrid analyzer quadrupole - time of flight (LC-QTOF). After analytical development, the method was validated according to do Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, evaluating linearity, limit of detection, limit of quantification, matrix effect, precision, intermediate precision, bias and dilution integrity, besides recovery and process efficiency. The developed method comprised MDA, MDMA, 2C-B, DOB, ketamine, mCPP, cocaine and cocaethylene determination. Real samples related to non-natural deaths were collected at Institute of the Legal Medicine Aristoclides Teixeira, Goiânia, Goiás, Brazil, and analyzed by the developed method. 56 cases were selected, most of them related to fire gun injury and traffic events, 28,5% (n=16) of them being positive for cocaine and/or cocaethylene. None of the other drugs comprised in the analysis were detected in these samples

Synthetic Cannabinoid-Induced Immunosuppression Augments Cerebellar Dysfunction in Tetanus-Toxin Treated Mice

Synthetic cannabinoids are one of most abused new psychoactive substances. The recreational use of abused drug has aroused serious concerns about the consequences of these drugs on infection. However, the effects of synthetic cannabinoid on resistance to tetanus toxin are not fully understood yet. In the present study, we aimed to determine if the administration of synthetic cannabinoids increase the susceptibility to tetanus toxin-induced motor behavioral deficit and functional changes in cerebellar neurons in mice. Furthermore, we measured T lymphocytes marker levels, such as CD8 and CD4 which against tetanus toxin. JWH-210 administration decreased expression levels of T cell activators including cluster of differentiation (CD) 3ε, CD3γ, CD74p31, and CD74p41. In addition, we demonstrated that JWH-210 induced motor impairment and decrement of vesicle-associated membrane proteins 2 levels in the cerebellum of mice treated with tetanus toxin. Furthermore, cerebellar glutamatergic neuronal homeostasis was hampered by JWH-210 administration, as evidenced by increased glutamate concentration levels in the cerebellum. These results suggest that JWH-210 may increase the vulnerability to tetanus toxin via the regulation of immune function.

Evaluation of the Abuse Potential of Novel Amphetamine Derivatives with Modifications on the Amine (NBNA) and Phenyl (EDA, PMEA, 2-APN) Sites

Recently, there has been a rise in the number of amphetamine derivatives that serve as substitutes for controlled substances (e.g. amphetamine and methamphetamine) on the global illegal drug market. These substances are capable of producing rewarding effects similar to their parent drug. In anticipation of the future rise of new and similar psychoactive substances, we designed and synthesized four novel amphetamine derivatives with N-benzyl, N-benzylamphetamine HCl (NBNA) substituent on the amine region, 1,4-dioxane ring, ethylenedioxy-amphetamine HCl (EDA), methyl, para-methylamphetamine HCl (PMEA), and naphthalene, 2-(aminopropyl) naphthalene HCl (2-APN) substituents on the phenyl site. Then, we evaluated their abuse potential in the conditioned place preference (CPP) test in mice and self-administration (SA) test in rats. We also investigated the psychostimulant properties of the novel drugs using the locomotor sensitization test in mice. Moreover, we performed qRT-PCR analyses to explore the effects of the novel drugs on the expression of D1 and D2 dopamine receptor genes in the striatum. NBNA, but not EDA, PMEA, and 2-APN, induced CPP and SA in rodents. None of the test drugs have produced locomotor sensitization. qRT-PCR analyses demonstrated that NBNA increased the expression of striatal D1 dopamine receptor genes. These data indicate that NBNA yields rewarding effects, suggesting potential for abuse. Continual observation for the rise of related substances is thus strongly encouraged.